A1 is induced by pathogen ligands to limit myeloid cell death and NLRP3 inflammasome activation

Abstract Programmed cell death pathways play an important role in innate immune responses to infection. Activation of intrinsic apoptosis promotes infected cell clearance; however, comparatively little is known about how this mode of cell death is regulated during infections and whether it can induce inflammation. Here, we identify that the pro‐survival BCL‐2 family member, A1, controls activation of the essential intrinsic apoptotic effectors BAX/BAK in macrophages and monocytes following bacterial lipopolysaccharide (LPS) sensing. We show that, due to its tight transcriptional and post‐translational regulation, A1 acts as a molecular rheostat to regulate BAX/BAK‐dependent apoptosis and the subsequent NLRP3 inflammasome‐dependent and inflammasome‐independent maturation of the inflammatory cytokine IL‐1β. Furthermore, induction of A1 expression in inflammatory monocytes limits cell death modalities and IL‐1β activation triggered by Neisseria gonorrhoeae‐derived outer membrane vesicles (NOMVs). Consequently, A1‐deficient mice exhibit heightened IL‐1β production in response to NOMV injection. These findings reveal that bacteria can induce A1 expression to delay myeloid cell death and inflammatory responses, which has implications for the development of host‐directed antimicrobial therapeutics.


Introduction
The programmed death of innate immune cells can be deployed to combat infection.In the case of bacteria, the cytosolic NOD-like receptor (NLR) and absent in melanoma 2 (AIM2) sensing of specific bacterial ligands, or the activity of effector molecules, leads to the assembly and activation of multimeric signalling platforms called inflammasomes.Inflammasomes, such as the NLRP3 inflammasome, activate caspase-1 to mature the pro-inflammatory cytokine, interleukin (IL)-1b, as well as to cleave and activate the pore-forming protein Gasdermin D (GSDMD) that can induce a lytic form of cell death called pyroptosis.GSDMD pores facilitate the release of inflammatory cytokines and damage-associated molecular patterns (DAMPs) that alert the host immune system, while pyroptosis restricts intracellular bacterial replication (Broz & Dixit, 2016).Caspase-11-sensing of the lipid A moiety of Gram-negative bacteriaderived LPS also results in GSDMD-mediated pyroptosis that promotes NLRP3 inflammasome activation via potassium ion (K + ) efflux (Kayagaki et al, 2015;Ruhl & Broz, 2015;Shi et al, 2015).While programmed cell death can protect mammals from infection, it can also limit the ability of phagocytes to eliminate pathogens in a cell-autonomous manner.Consistent with this, many pathogens have evolved virulence factors that manipulate host cell death machinery in order to evade antimicrobial responses (Cunha & Zamboni, 2013;Naderer & Fulcher, 2018;Eng et al, 2020).This includes intracellular pathogens (e.g., Legionella) that evade detection by cytosolic sensors and delay macrophage cell death in order to establish a replicative niche (Speir et al, 2016).
While mitochondrial apoptosis is generally considered immunologically silent, recent reports have suggested that apoptotic factors and/or mitochondria promote NLRP3 inflammasome activity.In fact, several models propose that mitochondrial damage is essential for NLRP3 inflammasome activation and assembly in response to canonical stimuli, such as the bacterial toxin nigericin or ATP (reviewed in Lawlor & Vince, 2014;Yabal et al, 2019;Riley & Tait, 2020).However, a number of these models have now been challenged (Allam et al, 2014;Dang et al, 2017).For instance, it was proposed that canonical NLRP3 triggers universally induce mitochondrial stress and the release of oxidised mitochondrial DNA (mtDNA) via the BAX/ BAK pore or the mitochondrial Permeability Transition Pore (mPTP) to activate NLRP3 (Shimada et al, 2012;Xian et al, 2022).Yet, even the original report by Shimada et al (2012) actually showed that the majority of the inflammasome response was dependent on the DNA sensor AIM2.Other recent studies have further contradicted these models by revealing that mtDNA depletion does not impact NLRP3 responses to ATP (Dang et al, 2017) and by showing genetically that BAX/BAK, caspase-9, and the mPTP component cyclophilin-D are not required for canonical NLRP3 activation (Allam et al, 2014;Vince et al, 2018).Despite debate surrounding the actual contribution of the mitochondria to canonical NLRP3 inflammasome activity, a number of studies have revealed that both intrinsic and extrinsic apoptotic cell death modalities can crosstalk with inflammatory signalling pathways, particularly the NLRP3 inflammasome (Lawlor et al, 2015;Conos et al, 2017;Malireddi et al, 2018;Vince et al, 2018).In the case of intrinsic apoptosis, BAX/BAK signalling in LPS-primed macrophages has been shown to trigger caspase-3/-7-dependent NLRP3 inflammasome activation, as well as caspase-8-mediated IL-1b cleavage (Chauhan et al, 2018;Vince et al, 2018).It has also become apparent that IL-1b and other inflammatory DAMPs released by apoptotic cells can activate the immune system during infection, akin to pyroptosis (Chauhan et al, 2018;Deo et al, 2020;Orzalli et al, 2021).However, further work is needed to delineate how BAX/ BAK activation is regulated during infection and whether these signals can fine-tune cell death and inflammatory responses in diverse innate immune cells.
Here, we investigate how bacterial sensing alters the ability of myeloid cells to induce mitochondrial apoptosis and inflammation.
We show that pro-survival protein, A1, is dynamically regulated in macrophages after LPS sensing.A1 acts as a key regulator to control the timing of apoptosis in macrophages upon combined loss of BCL-XL and MCL-1.Moreover, for the first time, we reveal that inflammatory monocyte survival depends primarily on A1 and MCL-1 following LPS exposure and, importantly, that A1 upregulation supresses pro-inflammatory IL-1b production in response to Neisseria gonorrhoeae-derived outer membrane vesicles (NOMVs) both in vitro and in vivo.

LPS priming delays BAX/BAK-mediated apoptosis and IL-1b release in macrophages
The pro-survival BCL-2 family members MCL-1 and BCL-XL are reportedly master regulators of intrinsic apoptosis in bone marrowderived macrophages (BMDMs) (Speir et al, 2016;Vince et al, 2018).Here, we confirm that wild-type (WT) BMDMs undergo rapid apoptotic cell death upon co-targeting of BCL-XL and MCL-1 using the BH3-mimetic  or the BCL-XL-selective inhibitor A-1331852 (852) in combination with either the MCL-1-specific inhibitor S63845 (S6) (Kotschy et al, 2016) or the protein synthesis inhibitor cycloheximide (CHX; to deplete MCL-1; Goodall et al, 2016) (Figs 1A and EV1A).In line with previous findings (Chauhan et al, 2018;Vince et al, 2018), we also observed that dual targeting of BCL-XL and MCL-1 with ABT-737/S63845, A-1331852/S63845, or ABT-737/CHX in LPS-primed macrophages induced cell death (Figs 1A and EV1A) and .Surprisingly, upon closer examination of BAX/BAK-mediated responses at 6 and 24 h, we observed that cell death induced by ABT-737 or A-1331852 in combination with S63845 was delayed at 6 h in LPS-primed cells compared to unprimed macrophages (Figs 1A and B and EV1A and B).Moreover, we observed that both cell death and IL-1b activation was delayed in LPS-primed macrophages in response to ABT-737/ S63845 or A-1331852/S63845 when compared to ABT-737/CHX treatment, despite efficient priming, as shown by equivalent TNF release (Figs 1A-D and EV1A-H).These findings suggest that, in this context, CHX prevents the early and transient upregulation of an additional pro-survival factor by LPS.
To identify the short-lived factor delaying cell death and inflammatory cytokine production, we performed 3 0 mRNA sequencing of LPS-treated versus untreated BMDMs.As expected, we observed significant global changes in gene signatures in BMDMs exposed to LPS (Fig EV1I).Moreover, gene ontology (GO) analyses largely revealed enrichment of processes involved in cytokine and signalling responses, host defence, and cell death regulation in response to LPS (Fig EV1J).Examination of a targeted list of significantly enriched cell death genes from our RNA-seq dataset revealed significant upregulation of genes involved in inflammasome/pyroptotic (e.g., Nlrp3, Aim2, Casp1, Gsdmd, Il1b, Il18) and extrinsic cell death (e.g., Fas, Tnfrsf1b, Birc3, Tnfaip3, Mlkl, Casp8, Cflar) signalling (Fig 1E).In comparison, we observed few significantly enriched genes related to intrinsic apoptotic signalling (e.g., downregulated Bcl2 and Bid expression) in response to LPS treatment, with upregulation of all 4 isoforms of the pro-survival BCL-2 family member, Bcl2a1 (A1), being the most obvious change at 7 h (Fig 1E).Further examination of pro-survival BCL-2 family expression over time in response to LPS revealed relatively stable expression of Bcl2l1 (encodes BCL-XL) and Mcl1 mRNA (Figs 1F and G,and EV1K).Aligning with the RNA-seq data, Bcl2a1 was absent in unstimulated BMDMs and rapidly, albeit transiently, induced at the mRNA and protein level in response to Escherichia coli O111:B4 (B4) and/or O55:B5 (B5) LPS serotypes (Figs 1F and G,and EV1K).In agreement with past reports that A1 is degraded by the proteasome (Kucharczak et al, 2005;Herold et al, 2006), we observed that the proteasome inhibitor MG132, but not the lysosome inhibitor Bafilomycin A1 (BafA1) nor the pan-caspase inhibitor Q-VD-OPh (QVD), impeded A1 turnover in CHX pulse-chase experiments performed in LPS-primed cells (Fig EV1L).As expected, turnover of short-lived MCL-1 and pro-IL-1b proteins (Vijayaraj et al, 2021) was also prevented by MG132 (Fig EV1L).Therefore, transient induction of A1 upon LPS priming may explain why apoptosis is delayed upon selective BCL-XL and MCL-1 targeting compared to ABT-737/CHX treatment, as CHX would rapidly deplete the pool of A1 protein.
Correlating with early apoptotic cell death in LPS/ABT-737/ S63845-treated A1-deficient BMDMs, we observed elevated bioactive IL-1b (p17) and inflammasome-associated caspase-1 (p20) over time, compared to more delayed secretion in WT cells (Figs 2B and E,and EV2C,D and G).TNF production (marker of inflammasome priming) was not influenced by A1 deficiency nor by BH3-mimetic treatment (Figs 2F and EV2E and F).Of note, while we did see some interexperiment variation in IL-1b levels detected in WT cell supernatants over time, this likely reflects variation in the magnitude and kinetics of LPS-induced A1 turnover between experiments, as well as when regulatory events that limit NLRP3 inflammasome activity and pro-IL-1b levels are induced (Yabal et al, 2019;Vijayaraj et al, 2021).Surprisingly, parallel testing of the BCL-2 inhibitor, ABT-199, also revealed flexible pro-survival protein requirements, as ABT-199/S63845 treatment also induced IL-1b production in LPS-primed A1-deficient BMDMs, albeit at lower levels than with ABT-737/S63845 (Fig EV2H and I).Importantly, irrespective of whether BCL-XL or BCL-2 were targeted alongside MCL-1, A1 upregulation always delayed early cell death and IL-1b release to BH3-mimetic treatment, which would represent the acute phase of an infectious response.
Mechanistically, and mirroring past reports in a WT setting (Vince et al, 2018), addition of the NLRP3 inhibitor MCC950 (950) (Coll et al, 2015) had no effect on cell death (Figs 2A and EV2A and B) but did block caspase-1 cleavage and reduce IL-1b activation in the absence of A1 in LPS/ABT-737/S63845-treated BMDMs (Figs 2B and E,and EV2C,D and G), indicating that IL-1b activation is partly inflammasome mediated.In contrast, A1-deficient BMDMs responded normally to canonical NLRP3 and noncanonical Caspase-11 inflammasome stimuli, as well as infection with the intracellular pathogen Salmonella enterica Typhimurium (S.Typhimurium) (Fig EV2J -P), indicating that A1 only regulates NLRP3 inflammasome activation downstream of BAX/BAKdependent apoptosis.
In line with the published BAX/BAK-signalling model in WT macrophages (Vince et al, 2018), enhanced IL-1b activation in A1-deficient BMDMs was associated with the rapid loss of extrinsic cellular inhibitor of apoptosis 1 (cIAP1) and X-linked IAP (XIAP) proteins, and cleavage of caspase-8 and caspase-3, which was not abrogated by MCC950 (Figs 2B and EV2G).Altogether, these results suggest that A1 upregulation delays BAX/BAK-mediated apoptosis and downstream inflammasome-associated-caspase-1-and caspase-8-mediated IL-1b activation in macrophages.
In the absence of apoptotic caspase activity, specific activation of BAX/BAK induces TNF-and RIPK3 kinase-dependent necroptotic signalling and NLRP3 inflammasome activation To confirm that BAX/BAK activation in LPS-primed BMDMs solely triggers apoptosis in the absence of A1, we next examined whether the pan-apoptotic caspase inhibitor Q-VD-OPh could block cell ▸ Figure 2. A1 deficiency leads to accelerated apoptotic cell death and inflammasome-dependent and -independent IL-1b activation in LPS-primed macrophages.
In non-haematopoietic cells, under caspase-deficient conditions, BAX/BAK-driven MOMP has been shown to trigger IAP loss and noncanonical NF-jB-associated TNF production (linked to cIAP loss) to induce necroptosis (Giampazolias et al, 2017).We therefore queried whether Q-VD-OPh/ABT-737/S63845 treatment in unprimed and LPS-primed WT and A1-deficient macrophages promoted TNFdriven necroptosis.In unprimed macrophages, we observed considerable TNF secretion specifically in response to Q-VD-OPh/S63845/ .Correspondingly, we observed gradual cell death that was reduced by TNF neutralisation in both WT and A1deficient BMDMs (Fig EV3D and E).In contrast, we observed no exaggeration of TNF secretion in LPS-primed WT cells upon caspase inhibition and BAX/BAK activation, however, we did observe enhanced TNF secretion in LPS-primed A1-deficient cells (Fig 3D and E) that display early MOMP (i.e., cytochrome loss).Importantly, we found that, again, TNF neutralisation attenuated necroptotic (Q-VD-OPh/S63845/ABT-737) but not apoptotic (S63845/ABT-737) cell death, as well as IL-1b activation in LPSprimed A1-deficient BMDMs (Figs 3F and G,and EV3F), albeit to a lesser extent than the RIPK3 inhibitor (Fig 3A, B and C).These data suggest that specific activation of BAX/BAK-dependent cell death can support TNF-driven necroptosis and IL-1b activation in LPSprimed macrophages when caspase activity is not fully attenuated.

A1 and MCL-1 prevent apoptosis and inflammatory signalling in LPS-primed monocytes
We often observed that loss of MCL-1 (S63845) and A1 activity could induce modest cleavage of caspase-8 (p43) and IL-1b secretion after LPS priming (Fig EV2C,D and G).We therefore considered the possibility that our BMDMs (derived using L929 cell-conditioned media (LCCM) that contains M-CSF) might be heterogeneous, akin to other bone marrow-derived myeloid cell models (Erlich et al, 2019), and contain a small percentage of immature monocyte/macrophages sensitive to A1 and MCL-1 loss.Consequently, we examined responses in day 4 LCCM cultures that predominantly contain CD11b + Ly6C + F4/80 lo-int inflammatory monocytes (henceforth referred to as BMMo) (Francke et al, 2011) Reminiscent of BMDMs (Figs 1G and EV1K), WT BMMo exhibited rapid and transient upregulation of A1 in response to LPS with ◀ Figure 3. A1 delays BAX/BAK-mediated necroptosis and NLRP3 inflammasome activation in LPS-primed macrophages.
Data information: Each dot represents an individual biological replicate.Data are representative of at least 3 independent biological experiments and presented as the mean + SEM.ns, not significant, *P < 0.05, ****P < 0.0001 (two-way ANOVA with Tukey's multiple comparisons test). (B) Ponceau stain was used as a control for the loading of total protein.
Source data are available online for this figure., which fits with a previous report that low XIAP levels in CD11b + Ly6C + F4/80 lo monocytes sensitises them to cIAP1/2-targeting with the Smac-mimetic, Birinapant (Rijal et al, 2018).Importantly, IL-1b activation was only partially blocked by MCC950, suggesting that both caspase-1 and caspase-8 contribute to IL-1b maturation in monocytic cells.Thus, A1 and MCL-1 act together to limit intrinsic apoptosis and pro-inflammatory IL-1b production in LPS-primed monocytes.

A1 deficiency sensitises monocytes to OMVs derived from Neisseria
Recently, a number of pathogens have been shown to inhibit host protein synthesis and deplete MCL-1, rendering cells sensitive to rapid killing upon BCL-XL loss/inactivation (Speir et al, 2016;Suzuki et al, 2018;Orzalli et al, 2021).In fact, outer membrane vesicles derived from the emerging superbug Neisseria gonorrhoeae (NOMVs) have been shown to cause mitochondrial dysfunction, leading to inhibition of host protein translation, and delayed BAX/ BAK-mediated cell death and NLRP3 inflammasome activation in macrophages (Deo et al, 2020).Thus, based on the specific requirement for A1 and MCL-1 in monocyte survival upon LPS exposure, we postulated that LPS contained within NOMVs might transiently upregulate A1 (and MCL-1) to delay BAX/BAK-mediated inflammasome activation in the short-lived inflammatory monocyte population (Yona et al, 2013) EV4E), suggesting pyroptotic signalling is triggered.Supporting this idea, we found that IL-1b secretion (and cell death) was comparable in Gsdmd À/À monocytes upon NOMV-induced apoptotic (NOMV) or necroptotic (QVD/ NOMV) signalling but IL-1b secretion was completely blocked in Gsdmd À/À monocytes when both pathways were inhibited (QVD/ 872/NOMV) (Fig EV4F -H).Collectively, these results suggest that, while A1 loss in inflammatory monocytes accelerates BAX/BAKmediated cell death and inflammatory signalling triggered by NOMVs, in the absence of apoptotic caspases this pathway can default to necroptosis and downstream NLRP3 inflammasome activation.Yet when both apoptosis and necroptosis are limited, NOMV-derived LPS may be sensed by caspase-11 to promote NLRP3 activity.It is important to note that, as these cells undergo MOMP (Ekert et al, 2004), they will die in a manner independent of apoptotic caspases, RIPK3, and inflammasome activity.

Enhanced IL-1b activity in A1-deficient mice to NOMV injection in vivo
Having established that A1 restricts NOMV-induced monocyte cell death and IL-1b activation in vitro, we next examined whether A1 upregulation could dampen the acute inflammatory response to N. gonorrhoeae extracellular infection in vivo.To do this, we injected WT and A1-deficient mice intraperitoneally with 100 lg of NOMVs and after 6 h harvested blood for ADVIA differential cell counts and flow cytometric analysis of leukocytes, and serum and peritoneal lavage fluid for cytokine analysis.Analysis of immune cells in the blood revealed that both the WT and A1 À/À NOMVtreated mice exhibited the lymphopenia characteristic of sepsis but showed no meaningful differences in immune cell subsets between genotypes at this time point (  EV5K).Similarly, serum and peritoneal fluid TNF and IL-6 levels were also largely equivalent between WT mice and A1-deficient mice, although A1 À/À mice did exhibit reduced levels of serum and EV5L and M).
Overall, these data demonstrate that A1 plays a systemic role in vivo in limiting inflammation arising from exposure to N. gonorrhoeaederived OMVs, which is not mediated solely via LPS-sensing.

Discussion
It is now becoming clear that apoptotic signalling can crosstalk with inflammatory cell death (e.g., pyroptosis) and promote NLRP3 inflammasome and IL-1b activation (Yabal et al, 2019;Speir & Lawlor, 2021).This work has centred around the activities of extrinsic apoptotic caspase-8, which, to date, include the regulation of transcriptional responses (Allam et  Loss of MCL-1 and BCL-XL (or BCL-2) activity in LPS-primed macrophages can trigger BAX/BAK-dependent inflammation associated with the loss of ripoptosome repressors XIAP and cIAP1, and through effector caspase-3/À7-mediated activation of caspase-8 to directly cleave IL-1b, and activation of NLRP3 via pannexin-1 channel-dependent K + efflux (Chauhan et al, 2018;Vince et al, 2018;Chen et al, 2019).One puzzling element to this proposed model has been the fact that LPS priming significantly delays BAX/ BAK-mediated cell death signalling induced by selective targeting of MCL-1 and BCL-XL (Vince et al, 2018).We now reveal the explanation for this discrepancy is the transient expression of the prosurvival protein A1, which not only limits cell death upon MCL-1 and BCL-XL targeting but also, unexpectedly, prevents the more protracted BAX/BAK inflammatory signalling observed upon MCL-1 and BCL-2 inhibition.Considering the emerging role for BCL-2 in regulating macrophage survival under pathogen-induced stress (Chauhan et al, 2018;Simpson et al, 2022), understanding how short-lived A1 promotes late-stage survival dependency on BCL-2 will be of interest.
Our attempts to verify that specific loss of MCL-1, BCL-XL, and A1 activity triggers apoptosis in macrophages uncovered that these cells are poised to die via necroptosis basally and upon TLR4 ligation.This contrasts with the block in cell death achieved with panapoptotic-caspase inhibition in LPS-primed BMDMs treated with ABT-737/CHX (Vince et al, 2018).In the case of ABT-737/CHX treatment, depletion of the LPS-inducible c-FLIP by CHX is proposed to promote a pro-apoptotic caspase-8 homodimeric complex that is more effectively inhibited by Q-VD-OPh (Maelfait et al, 2008;Wang et al, 2008;Allam et al, 2014;Brumatti et al, 2016).Why necroptosis occurs in a proportion of LPS/ABT-737/S68345-treated A1-deficient BMDMs upon caspase inhibition is less clear and warrants future investigation.It is tempting to speculate that, due to ineffective caspase-8 inhibition in these cells (Brumatti et al, 2016;Simpson et al, 2022), a threshold of c-FLIP expression and/or reduced IAP levels predisposes them to necroptosis through TNF signalling.Overall, this work shows that, while A1, MCL-1, and BCL-XL act chiefly to limit intrinsic apoptosis in LPS-primed macrophages, these cells are capable of engaging other cell death modalities, akin to the flexibility discovered during extrinsic cell death signalling in innate and nonimmune cells (Newton et al, 2014(Newton et al, , 2019b;;Fritsch et al, 2019;Doerflinger et al, 2020;Malireddi et al, 2020;Simpson et al, 2022).
We found that A1 was strongly but transiently induced by LPS in bone marrow monocytes, akin to macrophages.However, BH3mimetic screening revealed a distinct pro-survival protein profile, with MCL-1 and A1 playing a vital role in limiting intrinsic apoptosis in inflammatory monocytes.Remarkably, loss of MCL-1 and A1 activity also triggered IL-1b activation, via caspase-8 and NLRP3 inflammasome activity, confirming that A1 delays BAX/ BAK-mediated inflammatory signalling in both monocytes and macrophages.Both A1 and MCL-1 have previously been implicated in limiting neutrophil cell death basally, upon activation and to death receptor FAS ligation (Dzhagalov et al, 2007;Vier et al, 2016;Csepregi et al, 2018;Schenk et al, 2020).However, despite these observations, and to our knowledge, this is the first report that A1 and MCL-1 regulate Ly6C hi monocyte turnover.It is worth acknowledging that, due to the embryonic lethality of MCL-1 knockout mice (Rinkenberger et al, 2000), most studies have examined monocyte homeostasis using mice with conditional deletion of Mcl-1 in myeloid cells (LysM Cre transgenic) that renders them neutropenic (Steimer et al, 2009;Csepregi et al, 2018).A potential caveat to these studies is the possibility that residual MCL-1 expression maintains monocyte numbers due to ineffective deletion, as seen previously with extrinsic regulators (Kang et al, 2013;Wong et al, 2014;Lawlor et al, 2015;Vince et al, 2018), and the potential for compensatory myelopoiesis or pro-survival protein expression (Dzhagalov et al, 2007).Overall, the critical need to maintain MCL-1 sufficiency is underscored by the relatively normal granulopoiesis observed in heterozygous MCL-1 mutant mice (Brinkmann et al, 2017), and the relative resistance of murine immune cells to S63845 (Vince et al, 2018;Rohner et al, 2020).Neisseria gonorrhoeae-derived outer membrane vesicles deliver LPS and a range of effector proteins and toxins to host cells (Pathirana & Kaparakis-Liaskos, 2016;Dhital et al, 2021), including the mitochondria-targeting PorB protein that disrupts mitochondrial homeostasis and induces MCL-1 depletion to trigger late-stage apoptosis in macrophages that we have shown can be amplified by BCL-XL targeting (Deo et al, 2018(Deo et al, , 2020)).Our data support a role for A1 (and MCL-1) in limiting NOMV-induced BAX/BAK-mediated apoptosis, necroptosis, and possibly pyroptosis in infiltrating inflammatory monocytes that act as bystander cells during extracellular infections.A1 expression also limited NOMV-induced IL-1b responses that were partly dependent on caspase-8 and NLRP3 inflammasome activity.Intriguingly, these results differ from a recent finding in macrophages that NOMV-induced BAK activation solely triggers K + efflux-dependent NLRP3 inflammasome responses (Deo et al, 2020).Consequently, our results highlight the variance in cell death signalling molecules engaged upon NOMV exposure in individual innate immune cells.This divergence is likely to play a vital role in infectious outcomes, where the rapid turnover and cell death plasticity in infiltrating inflammatory Ly6C hi monocytes will be key to driving acute inflammation, while delayed cell death in longer-lived macrophages may preserve the replicative niche.Impressively, A1 expression reduced sepsis-induced IL-1b activation in mice exposed to NOMVs despite us failing to observe any depletion of innate immune cells and/or signs of local inflammation.Therefore, it remains to be seen whether over time A1 expression impacts select populations.Moreover, it is unclear whether A1 upregulation is beneficial or deleterious for the host during infection, since this may be multi-factorial depending on the pathogen (Gangoda et al, 2021), cell types involved, and the role IL-1b plays in shaping the immune response.Nevertheless, with a growing number of pathogens reported to deplete MCL-1 and target BCL-XL or BCL-2, and reports defining the potential use of BH3-mimetic drugs as antimicrobials (Ohmer et al, 2016;Speir et al, 2016;Chauhan et al, 2018;Suzuki et al, 2018;Deo et al, 2020;Inde et al, 2021;Orzalli et al, 2021), it will be vital to assess the role of A1 in infectious disease, particularly as it is conceivable that anticancer A1/BFL-1 inhibitors in development (Li et al, 2021) could be re-purposed as antimicrobial therapeutics.

Mice
All mice were housed under standard conditions at the Walter + Eliza Hall Institute of Medical Research (WEHI), Australia, and Monash University, Australia.All procedures were performed in accordance with the National Health and Medical Research Council Australian Code of Practice for the Care and Use of Animals and approved by the WEHI (2017WEHI ( .026, 2020.038) .038)or Monash Animal Ethics Committees (MARP/2016/099, MMCB/2019/28-BC, MMCB/2021/25).Male and female C57BL/6 wild-type (WT), A1-deficient (A1 À/À ) (Schenk et al, 2017) and GSDMD-deficient (Kayagaki et al, 2015) mice at > 6 weeks of age were used for in vitro generation of bone marrowderived macrophages (BMDMs) or monocytes.For in vivo experiments, 8-12-week-old male WT and A1 À/À mice were injected intraperitoneally with 100 lg of NOMVs or 100 lg LPS.Mice were euthanised after 5-6 h (h) and cardiac bled for FACS analysis, differential blood counts (ADVIA 120 cell analyser, Bayer Diagnostics), and serum isolation.Peritoneal lavages were performed using ice cold 5 mM EDTA in PBS and fluid collected following centrifugation.Serum and peritoneal lavage fluid were stored at À80°C prior to cytokine analysis by ELISA and immunoblot.
Purification of outer membrane vesicles (OMVs) from N. gonorrhoeae The N. gonorrhoeae MS11-A strain was cultured in gonococcal (GC) liquid media supplemented with Deakin modified isovitalex (DMIV) and 0.01 M NaHCO 3 at 37°C, shaking at 200 rpm.Bacterial cultures were grown to an optical density (OD 600 ) of ~0.5 (mid-log phase), diluted in fresh media (1:1,000 dilution), and incubated until an OD 600 ~0.8 was reached.Bacteria were removed by centrifugation (12,000 × g for 12 min at 4°C), and the spent culture medium was harvested and filtered (using 0.45 lm nitrocellulose MF-Millipore TM membrane filters) before NOMVs were isolated by density gradient ultracentrifugation, as detailed in (Deo et al, 2018).NOMVs were quantified using a bicinchoninic acid assay (Pierce BCA protein assay kit, Thermo Scientific CST #23225).

Salmonella infection
Salmonella enterica Typhimurium type strain SL1344 was plated onto Luria Bertani (LB) agar containing 50 lg/ml streptomycin (Sigma) and incubated at 37°C overnight.Single colonies were inoculated into 10 ml LB broth (+50 lg/ml streptomycin) and incubated in a 37°C shaking incubator at ~200 rpm for 16 h, then sub-cultured 1/50 in 5 ml LB broth, as above.Bacterial cell numbers were determined by absorbance at OD 600 and standardised to a multiplicity of infection (MOI) of 10 in un-supplemented DMEM.The infection protocol used is described in detail in (Ingle et al, 2021).Briefly, BMDMs were infected with SL1344 (or DMEM alone) at MOI:10 and centrifuged at 525 × g to synchronise bacterial uptake, then incubated at 37°C in 5% CO 2 for 30 min.Infective media was removed, and cells were washed and incubated with DMEM containing 100 lg/ml gentamicin for 1 h to inhibit extracellular bacterial growth.Finally, this media was replaced with DMEM containing 20% LCCM, 10% FBS, and 10 lg/ml gentamicin for the remainder of the experiment.Cell supernatants were harvested at 0, 3, 9 and 24 h post-infection (h.p.i.) for cytokine and LDH release (i.e., cell death).To quantify intracellular bacteria, the cell culture media was removed and BMDMs were washed 2× with warm PBS to remove extracellular bacteria, then lysed in 0.1% Triton X-100 by repetitive pipetting.Intracellular lysates were then serially diluted in PBS in duplicate, plated onto LB agar (+50 lg/ml streptomycin), and incubated overnight at 37°C.

Caspase activity assay
To assess caspase-3 activity, 1 × 10 5 BMDMs were plated per well in 96-well flat-bottom tissue culture-treated plates (BD Falcon), primed with LPS (50 ng/ml) for 3 h and then treated, as indicated with Q-VD-OPh (40 lM), ABT-737 (500 nM) and S63845 (10 lM).All stimulations were staggered in a reverse time course fashion to ensure identical LPS priming and QVD incubation periods across treatments.At experimental endpoint, supernatants were discarded after cell centrifugation and BMDMs lysed in 70 ll DISC lysis buffer (20 mM Tris, 150 mM NaCl, 2 mM EDTA, 1% TritonX-100, 10% Glycerol, H 2 O) for 1 h at room temperature with rotation.10 ll of cell lysate was used for protein quantification (Pierce TM BCA Protein Assay Kit, Thermo Fisher Scientific) according to manufacturer's instruction, and 50 ll was transferred to an opaque-walled, clear bottom 96-well flat bottom plate.DEVDase substrate (Ac-DEVD-AMC, BD Pharmingen TM Cat) was prepared to working concentration (20 lM) in assay buffer (20 mM HEPES pH 7.5, 10% Glycerol, 2 mM Dithiothreitol (DTT)).200 ll of prepared DEVDase substrate reagent was added to each well containing lysates and allowed to incubate overnight with rotation in the absence of light.After incubation, caspase AMC fluorescence was detected using a CLARIOstar Plus (BMG Labtech).Protein concentrations obtained from the BCA were used to normalise the AMC fluorescence intensity across samples.

Cytochrome-c release assay
Cytochrome-c release was measured under relevant specified conditions (see figure legends for details), as performed previously (Waterhouse & Trapani, 2003).Briefly, either unstained BMMo or macrophages, or Ly6C + -stained monocytes were washed 1× in PBS and resuspended in 50 ll MELB buffer (20 mM HEPES pH 7.5, 100 mM sucrose, 2.5 mM MgCl 2 , 100 mM KCl) supplemented with 0.025% w/v digitonin and complete protease inhibitors (Roche) for 10 min on ice to allow permeabilisation of the plasma membrane.Cells were then washed with MELB buffer before fixation in 50 ll eBioscience fixation buffer for 30 min on ice.Cells were washed 1× with eBioscience permeabilization buffer before incubation with an APC-conjugated anti-cytochrome-c antibody (REA702; Miltenyi Biotech) or APC-conjugated isotype control (REA293; Miltenyi Biotech) for 30 min on ice.Cells were washed 1× before data acquisition on a BD LSR-Fortessa X20 instrument and analysis using FlowJo.

Live-Cell imaging
To assess the kinetics and modes of cell death induced in LPSprimed BMDMs in response to BH3-mimetics, 5 × 10 4 macrophages were seeded into wells of a 96-well Thermo Scientific TM Nunc TM Edge 2.0 plate.After an overnight incubation to allow cell adhesion, culture medium was replaced with fresh DMEM (supplemented with 20% LCCM, 4 mM L-glutamine, 1 mM sodium pyruvate, and 100 U/ml penicillin/streptomycin) containing PI (200 ng/ml), SPY505-DNA (1×, Spirochrome) and, where indicated, LPS (50 ng/ ml).After 2.5 h cells were pre-treated, as indicated, with Q-VD-OPh (40 lM, In Vitro Technologies), MCC950 (5 lM, CP-456773 sodium salt, PZ0280), and/or GSK'872 (5 lM, MedChemExpress), rat antimouse TNF (XT-22 20 lg/ml) or rat anti-mouse IgG1 (GL113 Isotype control) monoclonal antibodies for 30 min prior to treatment with ABT-737 (500 nM) and S63845 (10 lM).To assess the kinetics of NOMV-induced monocyte cell death, sorted Ly6C hi monocytes were labelled with 100 nM Cell Tracker Green (CTG) (Invitrogen) for 10 min at 37°C in serum-free DMEM.Cells were then washed and seeded at 0.5-1 × 10 5 cells/well in 96-well flat bottom tissue culture-treated plates (Greiner).Monocyte cultures were pre-treated, as indicated, for 15-30 min with Q-VD-OPh (40 lM), MCC950 (5 lM), and GSK'872 (1 lM), prior to treatment with 50 lg/ml of NOMVs.Monocytes were then exposed to 200 ng/ml PI and imaged every 30 min on an IncuCyteâ S3 or SX5 Live-Cell Analysis System (Sartorius) at 10× magnification for up to 14 h.BMDM and monocyte images were analysed, and the % of dead cells was calculated by dividing the number of PI-positive cells by the total cell number (based on CTG + or SPY505-DNA + cells), as quantified by the IncuCyteâ Analysis Software.
RNA sequencing preparation and analysis 3 0 mRNA sequencing of LPS-treated versus untreated BMDMs was performed as described in (Simpson et al, 2022).Total RNA was extracted using the ISOLATE II RNA Mini Kit (Meridian Bioscience).The extracted RNA was analysed on the Agilent 4200 Tapestation prior to library preparation.3 0 mRNA-sequencing libraries were prepared using the QuantSeq 3 0 mRNA-Seq Library Prep (Lexogen) according to the manufacturer's instructions and sequenced on the NextSeq 500 (Illumina).The single-end 75 bp were demultiplexed using Casavav1.8.2.Cutadapt (v1.9) was used for read trimming (Martin, 2011).The trimmed reads were subsequently mapped to the mouse genome (mm10) using HISAT2 (Kim et al, 2019).FeatureCounts from Rsubread package (version 1.34.7) was used for read counting after which genes with less than 2 counts per million reads (CPM) in at least 3 samples were excluded from downstream analysis (Liao et al, 2014(Liao et al, , 2019)).Count data were normalised using the trimmed mean of M values (TMM) method and differential gene expression analysis was performed using the limma-voom pipeline (limma version 3.40.6)(Robinson & Oshlack, 2010;Law et al, 2014;Liao et al, 2014).Gene ontology (GO) analysis was performed using Metascape.Heatmaps were generated using pheatmap (version 1.0.12)(https://rdrr.io/cran/pheatmap/).The datasets generated during this study are available at GEO: GSE214525.
Quantitative RT-PCR 2 × 10 6 BMDMs were treated with 50 ng/ml LPS for up to 24 h before total RNA was extracted and purified using the Isolate II RNA mini kit (Bioline) or RNeasy Mini kit (Qiagen).RNA concentrations were quantified and standardised using a Nanodrop TM spectrophotometer.RNA (1 lg) was transcribed using the high-capacity cDNA reverse transcription kit (Applied Biosystems).The cDNA was then diluted 1:10 and qRT-PCR was performed on a QuantStudio 6 Flex PCR system with 2 ll of diluted cDNA, SYBR green reagent (Applied Biosystems), and the primer pairs listed below.Relative mRNA levels were calculated using the comparative delta-delta C t (2 ÀDDCt ) method (2 À [(dCt LPS given hour) À (dCT LPS 0 h)]) after gene expression was normalised to an internal house-keeping reference gene 18S.Results are displayed as the relative fold of mRNA levels compared to no LPS (0 h) treatment.Primer sequences used to assay relevant murine gene expression are as follows: 18S (For: 5 0 gtaacccgttgaaccccatt Rev: 3 0 ccatccaatcggtagtagcg) Bcl2a1 (For: 5 0 tccacaagagcagattgccctg Rev: 3 0 gccagccagatttgggttcaaac) Mcl1 (For: 5 0 agcttcatcgaaccattagcagaa Rev: 3 0 ccttctaggtcctgtacgtgga) Bcl2l1 (For: 5 0 gccacctatctgaatgaccacc Rev: 3 0 aggaaccagcggttgaagcgc).The specificity of each primer set was confirmed by the observation of a single peak in the melt curve graph of each qPCR run.
Cytokine analysis in supernatants and serum IL-1b (R&D), TNF (eBioscience), and IL-6 (eBioscience) ELISA kits were used according to the manufacturers' instructions.For detection of TNF in cell supernatants, or IL-6 in serum, samples were diluted 1:10 in assay diluent.

LDH assay
Supernatants were removed from cells and pelleted at 1,500 rpm to remove any debris.Control wells were left untreated or lysed in 1% Triton X-100 (Tx, Sigma-Aldrich, T9284) to establish maximum LDH release.% LDH activity was analysed using the Cytotoxicity Detection Kit (LDH), according to the manufacturer's instructions (Roche; 11644793001).Absorbance was measured at 490 nm and % cytotoxicity calculated as: [(experimental value À untreated control)/(Tx lysed control À untreated control)].

Statistical analysis
Flow cytometric data were analysed using FlowJo version 10 software.Error bars (SEM or SD) were calculated using Prism 8.1, as indicated in the figure legends.The number of times each experiment was repeated, and the number of animals used per experiment, are detailed in the figure legends.For in vitro analyses or in vivo analyses, a one-way or two-way ANOVA with Tukey's multiple comparisons test was performed to calculate significance.Normal distribution with similar variance between groups was assumed.For in vivo analyses of serum cytokines between 2 groups, an unpaired, two-tailed Student's t-test was performed to calculate statistical significance.No samples were excluded from analyses and no statistical methods were used to calculate sample size.

Figure 1 .
Figure 1.LPS-primed macrophages exhibit delayed sensitivity to BH3-mimetics.A-D Wild-type (WT) BMDMs were primed, as specified, with B5 LPS (50 ng/ml) for 3 h before addition of BH3-mimetics S63845 (S6; 10 lM), ABT-737 (737; 500 nM), and/or cycloheximide (CHX; 20 lg/ml) for 6 or 24 h, as indicated.(A, B) Cell death was measured by flow cytometric analysis of PI uptake.(C, D) IL-1b levels in the cell supernatants were measured by ELISA.E Heatmap showing significant differentially expressed cell death-associated genes (derived from apoptosis-related GO pathways) for untreated (UNT) and LPStreated (50 ng/ml) BMDMs at 7 h.Adjusted P ≤ 0.05 and cut-off values logFC ≥ 1 or logFC ≤ À1.F WT BMDMs were treated with LPS (50 ng/ml) for up to 24 h and mRNA transcript levels of Bcl2l1 (BCL-XL), Mcl1 (MCL-1), and Bcl2a1 (A1) were examined by qPCR.G WT BMDMs were treated with 50 ng/ml of either B4 or B5 LPS for up to 24 h and cell lysates were interrogated by immunoblot for the indicated proteins.Data information: Each dot represents an individual biological replicate.(A-D, F) Data are representative of at least three independent (A-D), 5 pooled (F) or at least two (G) biological experiments and (A-D, F) presented as the mean + SEM.ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001 (one-way ANOVA with Tukey's multiple comparisons test).(E) Data represent three individual biological replicates per condition.(G) Ponceau stain was used as a control for the loading of total protein.Source data are available online for this figure.
Data information: Each dot represents an individual (A, D-F) or the mean of three (C) biological replicates.Data are representative of at least three (A-C, E, F) or two (D) independent biological experiments and presented as the mean + or AE SEM.ns, not significant, ****P < 0.0001 (A, D-F two-way ANOVA with Tukey's multiple comparisons test) and (C unpaired two-tailed Student's t-test on the AUC).(B) b-actin was used as a control for the loading of total protein.Source data are available online for this figure.
(E) TNF and (G) IL-1b levels were measured in the cell supernatants by ELISA.(F) Cell death was measured by PI uptake via IncuCyte timelapse imaging.Data information: Each dot represents an individual (A, C-E, G) or the mean of at least five (B, F) biological replicates.Data are representative of two pooled biological experiments and presented as the mean + or AE SEM.ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001 (two-way ANOVA with Tukey's multiple comparisons test).Source data are available online for this figure.

Figure 5 .
Figure 5. A1 restricts IL-1b production and cell death in primary Ly6C hi monocytes treated with Neisseria gonorrhoeae-derived OMVs (NOMVs).AWT BMMo were treated with NOMVs for up to 18 h and cell lysates were interrogated for relevant proteins by immunoblot, as indicated.B-D WT and A1-deficient (A1 À/À ) BMMo were pre-treated, as indicated, with MCC950 (950) (5 lM) for 20-30 min and cultured with 50 lg/ml of NOMVs for 18 h.(B) IL-1b and (C) TNF levels were measured in the cell supernatants by ELISA.(D) Cell supernatants and lysates were analysed by immunoblot for specified proteins.E Primary Ly6C hi sorted monocytes from WT mice were cultured with 50 lg/ml of NOMVs for up to 5 h.Cell lysates were interrogated by immunoblot for specified proteins.F-I Primary Ly6C hi sorted monocytes from WT and A1-deficient mice were pre-treated with MCC950 (950) (5 lM), Q-VD-OPh (QVD) (40 lM), or GSK'872 (872) (1 lM) for 15-30 min, as indicated, and cultured with 50 lg/ml of NOMVs for up to 12 h.(F) Cell death was quantified by time-lapse live cell imaging of PI uptake in cell tracker green (CTG)-labelled monocytes.(G) IL-1b and (H) TNF levels in the cell supernatants were measured by ELISA at 5 h.(I) Cell supernatants and lysates were analysed by immunoblot for specified proteins at 5-6 h.Data information: Each dot represents an individual biological replicate (B, C, G, H).Data are representative of at least 3 independent (B, C) or 4 pooled (G, H) biological experiments and presented as the mean + SEM.ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001 (two-way ANOVA with Tukey's multiple compari- sons test).(F) Each dot represents the mean of n = 2 biological replicates per condition and are representative of 3 independent biological experiments and presented as the mean + SD. (A, D, E, I) Data are representative of at least two biological experiments, Ponceau stain or b-actin was used as a control for the loading of total protein and freshly harvested BMMo lysates were used as the control in (D) to avoid spontaneous cell death in culture.Source data are available online for this figure.
Figs 6A and B, and EV5A-F and Appendix Fig S5).Pro-inflammatory cytokine analysis revealed that serum IL-1b was significantly increased in NOMV-challenged A1 À/À mice compared to WT (Fig 6C), while serum TNF (Fig 6D) and IL-6 (Fig EV5G), as well as cytokine levels in the local peritoneal lavage fluid were equivalent (Fig EV5H-J).Crucially, serum immunoblot analysis revealed that, although IL-1b was mostly released in its immature pro-form, bioactive IL-1b (p17) was particularly evident in NOMV-injected A1-deficient mice (Fig 6E).In contrast to NOMVinduced responses, IL-1b levels in the serum and peritoneal lavage fluid were not perturbed in A1-deficient mice following low-dose LPS challenge (Figs 6F and

Figure 6 .
Figure 6.A1-deficient mice display heightened IL-1b levels in response to Neisseria gonorrhoeae-derived OMVs.A-E WT and A1-deficient mice were injected intraperitoneally with 100 lg of NOMVs or PBS.At 6 h, mice were sacrificed, and blood was collected for analysis.(A) Peripheral white blood cell (WBC) counts or (B) % WBC were measured using an ADVIA instrument.(C) IL-1b and (D) TNF levels were measured in the serum by ELISA.(E) Serum IL-1b activity (p17) was assessed by immunoblot.F-H WT and A1-deficient mice were injected intraperitoneally with 100 lg of LPS.At 6 h, mice were sacrificed, and blood was collected for analysis.(F) IL-1b, (G) ΤΝF and (H) IL-6 levels were measured in the serum by ELISA.Data information: Each dot represents an individual biological replicate.Data are representative of one (A, B) or at least 2 pooled (C, D, F-H) biological experiments and presented as the mean AE SEM.ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001 (A-D one-way ANOVA with Tukey's multiple comparisons test) and (F-H unpaired, two tailed Student's t-test).(E) Immunoblot of n = 4 individual biological replicates per genotype, Ponceau stain was used as a control for the loading of total protein.Source data are available online for this figure.